A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.
Objective: Our objective was to examine if a high animal protein intake from milk or meat increased s-insulin and insulin resistance in healthy, prepubertal children. A high animal protein intake results in higher serum branched chain amino acids (BCAA; leucine, isoleucine and valine) concentrations, which are suggested to stimulate insulin secretion. Furthermore, milk possesses some postprandial insulinotrophic effect that is not related to its carbohydrate content. Design: A total of 24 8-y-old boys were asked to take 53 g protein as milk or meat daily. At baseline and after 7 days, diet was registered, and insulin, glucose, and amino acids were determined. Insulin resistance and beta cell function were calculated with the homeostasis model assessment. Results: Protein intake increased by 61 and 54% in the milk-and meat-group, respectively. In the milk-group, fasting s-insulin concentrations doubled, which caused the insulin resistance to increase similarly. In the meat-group, there was no increase in insulin and insulin resistance. As the BCAAs increased similarly in both groups, stimulation of insulin secretion through BCAAs is not supported. Conclusions: Our results indicate that a short-term high milk, but not meat, intake increased insulin secretion and resistance. The long-term consequences of this are unknown. The effect of high protein intakes from different sources on glucose-insulin metabolism needs further studying.
Hypoallergenic milk formulas are used as an alternative diet for infants who have allergies to cow's milk when breast-feeding is not possible. These products are based on proteins, which have been heat-treated and hydrolyzed to a different degree in order to cleave antibody-binding structures. Even extensively hydrolyzed products have occasionally been observed to elicit allergic reactions in sensitized infants, however. Therefore, the parameters of relevance to allergenic potential require more investigation. The objective of the present study was to investigate 12 different hydrolyzed milk formulas for their contents of potentially allergenic protein material, i.e. material that may induce allergenicity or elicit allergic responses in already sensitized individuals. Analytical methods applied were gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE, immunoblotting, dot-immunobinding, and ELISA. Care was taken to assure that all protein fractions were investigated, including supernatants and precipitates following centrifugation of the milk formulas. By gel filtration, protein material with apparent molecular masses of 7 to >30 kDa was detected. Analysis by SDS-PAGE of formula precipitates showed that proteins with a molecular mass above 20 kDa were present even in some of the extensively hydrolyzed formulas. Residual antigenic beta-lactoglobulin was found by ELISA in all products. By immunoblotting and dot-immunobinding with antibodies against total whey, caseins, or Kunitz soybean trypsin inhibitor, we observed antigenic material mainly in partially hydrolyzed products. We concluded that SDS-PAGE of formula supernatants and precipitates gave the most differentiated profile of hydrolyzed formulas and that this method is well suited for screening potential allergenicity.
The plasma membrane of the cereal aleurone layer is the site of perception of germination signals and release of enzymes to the starchy endosperm. Analysis of membrane proteins is challenging due to their hydrophobicity and low abundance; thus, little is known about the membrane proteins involved in seed germination. A membrane fraction highly enriched for the plasma membrane H+-ATPase was prepared from barley aleurone layers by aqueous two-phase partitioning. Because detergent and salt washes did not efficiently remove soluble proteins from the membrane preparations, an alternative procedure was developed, comprising batch reversed-phase chromatography with stepwise elution of hydrophobic proteins by 2-propanol. Proteins in the most hydrophobic fraction were separated by SDS-PAGE and identified by LC-MS/MS and barley EST sequence database search. The method was efficient for enrichment of integral membrane proteins with relatively low levels of soluble contaminating proteins. Forty-six proteins associated with barley aleurone plasma membranes were identified, including proteins with more than 10 transmembrane domains. Among the identified proteins were two new isoforms of the plasma membrane H+-ATPase, two proteins possibly involved in ion-channel regulation, and two proteins of unknown function. This represents the first analysis of membrane proteins involved in seed germination using a proteomics approach.
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